Project Summary Cell movement through three-dimensional (3D) extracellular matrix (ECM) is an essential component of normal physiology and disease, including wound healing and tumor metastasis. Understanding how cells move through structurally diverse 3D matrices will be essential to design therapies aimed at controlling cell migration in the body. During 3D migration, both metastatic tumor cells and wound healing fibroblasts are faced with the same problem: how to efficiently move the bulky, stiff nucleus. While the power of actomyosin contractility is essential for cells to move their nuclei in 3D matrices, it is not understood how it is regulated by the ECM structure or physically coupled to the nucleus. An additional layer of complexity comes from the fact that the 3D matrix structure can govern actomyosin contractility to dictate the type of protrusions cells use to move (i.e. migratory plasticity). By understanding how the structure of the 3D ECM affects the physical properties of the nucleus and actomyosin contractility, we aim to create a conceptual framework to explain how and why human cells switch between distinct 3D migration mechanisms. We recently discovered that human cells moving in a linearly elastic 3D matrix rely on integrin-based cell-matrix adhesions and the power of actomyosin contractility to pull the nucleus forward, like a piston, and switch from using low-pressure lamellipodia to high-pressure lobopodial protrusions. This project will test the hypothesis that mechanical stress on the nucleus reprograms intracellular architecture and polarity to power the nucleus, and thereby the cell through 3D matrices. To achieve these goals, we will combine biophysical and cell biology approaches to measure mechanical stress on the nucleus and determine the molecular connections between discreet cytoskeletal elements required for high-pressure 3D motility. Aim 1 will manipulate the physical structures of the matrix and the nucleus and measure the ability of the cells to assemble and activate the nuclear piston mechanism. This approach will establish why the 3D matrix can reprogram cellular force production to switch cells to pressure-driven 3D migration. Aim 2 will identify the actomyosin machinery that is specifically activated by 3D matrix-cell interactions to generate pressure and govern migratory plasticity. These experiments will clearly distinguish the actomyosin filaments responsible for pulling the nuclear piston forward from those that respond to matrix stiffness by increasing traction force. Aim 3 will determine the mechanisms by which vimentin intermediate filaments transmit force to the nucleus to sustain intracellular polarization and directional cell movement in the narrow confines of the 3D matrix. These novel approaches will determine if the nucleus is a mechanosensor that responds to 3D matrix structure by governing actomyosin contractility and the mode of 3D cell migration. This enhanced understanding of the fundamental principles of directional 3D cell motility and migratory plasticity will lead to new therapeutic strategies to control normal and abnormal cell movement in the body.